Centromeres of eukaryotic chromosomes are specific regions along the chromatin fiber that play a fundamental role in chromosome movement during cell division. Centromere DNA sequences isolated from the yeast, Saccharomyces cerevisiae, enable foreign DNA introduced into yeast to function as ordinary yeast chromosomes during cell division. We will examine how the centromere DNA sequence interacts with chromatin components in the cell nucleus, including nuclear proteins and microtubules, to give rise to a functional centromeric unit. We will map the location of nuclease cleavage sites and will measure the coiling of the DNA duplex within centromeric chromatin to determine the organization of centromere DNA, histones and nonhistone proteins. The proteins that are associated with centromere DNA in vivo will be identified by direct isolation of intracellular centromere plasmid-protein complexes. The bound proteins will be characterized in terms of their DNA-binding properties by a filter binding assay and for their molecular heterogeneity by gel electrophoresis. We will then examine how these proteins interact with centromere DNA and microtubules to give rise to a functional centromeric unit. With the isolation of centromere specific DNA-binding proteins we should eventually be able to reconstitute the centromere spindle-fiber complex in vitro. Knowledge of the factors that control the organization of the centromere may provide fundamental principles governing the molecular mechanisms of cell division. Cell division is a central process of living organisms. Changes in the control of cell division may be an important mechanism that results in the production of aberrant cell populations, including cancer cells. Centromeric abnormalities, premature centromere separation and chromosome instability have been found in cases of Roberts' - SC phocomelia, ataxia telangiectasia, and a number of other syndromes. An understanding of the factors controlling cell division may be fundamental for the further understanding of these dysfunctions.